CN106381283A - Adipogenesis induction culture medium and adipogenic differentiation method - Google Patents
Adipogenesis induction culture medium and adipogenic differentiation method Download PDFInfo
- Publication number
- CN106381283A CN106381283A CN201610906762.7A CN201610906762A CN106381283A CN 106381283 A CN106381283 A CN 106381283A CN 201610906762 A CN201610906762 A CN 201610906762A CN 106381283 A CN106381283 A CN 106381283A
- Authority
- CN
- China
- Prior art keywords
- culture medium
- cell
- stem cell
- adipogenic induction
- induction culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000006698 induction Effects 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 27
- 239000001963 growth medium Substances 0.000 title claims abstract description 24
- 230000009815 adipogenic differentiation Effects 0.000 title abstract 6
- 230000011759 adipose tissue development Effects 0.000 title abstract 3
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims abstract description 34
- YASAKCUCGLMORW-UHFFFAOYSA-N Rosiglitazone Chemical compound C=1C=CC=NC=1N(C)CCOC(C=C1)=CC=C1CC1SC(=O)NC1=O YASAKCUCGLMORW-UHFFFAOYSA-N 0.000 claims abstract description 30
- CGIGDMFJXJATDK-UHFFFAOYSA-N indomethacin Chemical compound CC1=C(CC(O)=O)C2=CC(OC)=CC=C2N1C(=O)C1=CC=C(Cl)C=C1 CGIGDMFJXJATDK-UHFFFAOYSA-N 0.000 claims abstract description 30
- 102000004877 Insulin Human genes 0.000 claims abstract description 17
- 108090001061 Insulin Proteins 0.000 claims abstract description 17
- 229940125396 insulin Drugs 0.000 claims abstract description 17
- APIXJSLKIYYUKG-UHFFFAOYSA-N 3 Isobutyl 1 methylxanthine Chemical compound O=C1N(C)C(=O)N(CC(C)C)C2=C1N=CN2 APIXJSLKIYYUKG-UHFFFAOYSA-N 0.000 claims abstract description 16
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 claims abstract description 16
- 229960003957 dexamethasone Drugs 0.000 claims abstract description 16
- 229960000905 indomethacin Drugs 0.000 claims abstract description 15
- 229960004586 rosiglitazone Drugs 0.000 claims abstract description 15
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims abstract description 14
- 230000001939 inductive effect Effects 0.000 claims abstract description 13
- 230000002293 adipogenic effect Effects 0.000 claims description 31
- 210000000130 stem cell Anatomy 0.000 claims description 27
- 230000004069 differentiation Effects 0.000 claims description 22
- 239000007640 basal medium Substances 0.000 claims description 12
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 9
- 238000011534 incubation Methods 0.000 claims description 6
- ICTZKEXYDDZZFP-SRVKXCTJSA-N Pro-Arg-Pro Chemical compound N([C@@H](CCCN=C(N)N)C(=O)N1[C@@H](CCC1)C(O)=O)C(=O)[C@@H]1CCCN1 ICTZKEXYDDZZFP-SRVKXCTJSA-N 0.000 claims description 4
- 210000003716 mesoderm Anatomy 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 3
- 150000002475 indoles Chemical class 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 12
- 239000000463 material Substances 0.000 abstract description 4
- 210000004623 platelet-rich plasma Anatomy 0.000 abstract description 4
- 238000000338 in vitro Methods 0.000 abstract description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 2
- 230000001900 immune effect Effects 0.000 abstract description 2
- 230000002195 synergetic effect Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 46
- 102100034452 Alternative prion protein Human genes 0.000 description 12
- 235000015097 nutrients Nutrition 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 210000001519 tissue Anatomy 0.000 description 12
- 239000007788 liquid Substances 0.000 description 10
- 239000006228 supernatant Substances 0.000 description 9
- 238000005119 centrifugation Methods 0.000 description 8
- 230000003239 periodontal effect Effects 0.000 description 8
- 102100022119 Lipoprotein lipase Human genes 0.000 description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 6
- 108010016731 PPAR gamma Proteins 0.000 description 6
- 210000004195 gingiva Anatomy 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 210000004261 periodontium Anatomy 0.000 description 6
- NPGIHFRTRXVWOY-UHFFFAOYSA-N Oil red O Chemical compound Cc1ccc(C)c(c1)N=Nc1cc(C)c(cc1C)N=Nc1c(O)ccc2ccccc12 NPGIHFRTRXVWOY-UHFFFAOYSA-N 0.000 description 5
- 102000000536 PPAR gamma Human genes 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 230000029087 digestion Effects 0.000 description 5
- 239000010410 layer Substances 0.000 description 5
- 239000004017 serum-free culture medium Substances 0.000 description 5
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 4
- 102100037241 Endoglin Human genes 0.000 description 4
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 4
- 101000881679 Homo sapiens Endoglin Proteins 0.000 description 4
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 4
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 4
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 4
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 4
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000009977 dual effect Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 210000000981 epithelium Anatomy 0.000 description 4
- 230000001172 regenerating effect Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 102100023126 Cell surface glycoprotein MUC18 Human genes 0.000 description 3
- 101000623903 Homo sapiens Cell surface glycoprotein MUC18 Proteins 0.000 description 3
- 229930182555 Penicillin Natural products 0.000 description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 3
- 210000000988 bone and bone Anatomy 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 210000004400 mucous membrane Anatomy 0.000 description 3
- 229940049954 penicillin Drugs 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000017423 tissue regeneration Effects 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 208000009693 Gingival Hyperplasia Diseases 0.000 description 2
- 102000006354 HLA-DR Antigens Human genes 0.000 description 2
- 108010058597 HLA-DR Antigens Proteins 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000002458 cell surface marker Substances 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000006071 cream Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 108010007093 dispase Proteins 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 210000000214 mouth Anatomy 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 208000028169 periodontal disease Diseases 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- KYWKJKVQLGANBH-UHFFFAOYSA-N 3-butyl-1-methyl-7h-purine-2,6-dione Chemical compound O=C1N(C)C(=O)N(CCCC)C2=C1NC=N2 KYWKJKVQLGANBH-UHFFFAOYSA-N 0.000 description 1
- 208000010266 Aggressive Periodontitis Diseases 0.000 description 1
- 101100298998 Caenorhabditis elegans pbs-3 gene Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000002734 Collagen Type VI Human genes 0.000 description 1
- 108010043741 Collagen Type VI Proteins 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 208000034619 Gingival inflammation Diseases 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 description 1
- 241001597008 Nomeidae Species 0.000 description 1
- 208000025157 Oral disease Diseases 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 101000859864 Rattus norvegicus Gamma-crystallin E Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 208000009596 Tooth Mobility Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 210000001728 clone cell Anatomy 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 208000030194 mouth disease Diseases 0.000 description 1
- 210000002200 mouth mucosa Anatomy 0.000 description 1
- 210000001982 neural crest cell Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 201000006727 periodontosis Diseases 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 231100000241 scar Toxicity 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 210000001988 somatic stem cell Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0653—Adipocytes; Adipose tissue
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/40—Nucleotides, nucleosides or bases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/33—Insulin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1361—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from dental pulp or dental follicle stem cells
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Rheumatology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the field of the biotechnology, in particular to an adipogenesis induction culture medium and an adipogenic differentiation method. The adipogenesis induction culture medium comprises 3-isobutyl-1-methylxanthine, insulin, indomethacin, dexamethasone, rosiglitazone, PRP (Platelet Rich Plasma) and a basic culture medium. The invention provides an adipogenic differentiation method which enables various inducing factors to be subjected to combined application to act on mesenchymal stem cells including GMSCs and like, has a synergistic effect and can effectively induce the adipogenic differentiation of the mesenchymal stem cells, and the adipogenic differentiation effect of the adipogenic differentiation method is obviously superior to that of an existing conventional induction method. The GMSCs autologous materials are abundant, are easy in expansion in vitro and can be used for autotransplantation so as to avoid immunological rejection reaction.
Description
Technical field
The present invention relates to biological technical field, particularly to a kind of adipogenic induction culture medium and one-tenth fat differentiation method.
Background technology
Periodontal disease is not only the main cause causing adult to lose tooth, or harm Human Oral Cavity or even whole body health
Main oral disease.Periodontosis is the infectious diseases under the chronic inflammation stimulation of periodontium, is the microorganism in bacterial plaque
The inflammation causing under corresponding microenvironment, its damage periodontium, the especially forfeiture of the destruction of alveolar bone and periodontium,
Ultimately result in tooth mobility come off and alveolar bone absorption.And the target of periodontal treatment seeks to treat periodontal disease, improve
Microenvironment regenerates new connective tissue attachment and supporting tissue again, completes alveolar bone, cementum and pericemental structure.Often
Periodontal regenerative method, such as guided tissue regeneration, root planing etc., such method is simple, but can not be complete
Realize feature and structural paradenlal tissue regeneration, there is certain limitation.The appearance of periodontal tissue engineering technology and development
Provide brand-new thinking and method for periodontal regenerative.
Gum belongs to periodontium, is attached to the periodontal mucous membrane screen separating lower section periodontium and space outerpace
Barrier.Gum includes epithelium and lamina propria two parts, and from developing source, epithelium is derived from primitive mouth epithelium, on oral mucosa
Skin-deep continuity；Lamina propria then develops mesenchyma, epithelium and the mesenchyma continuous phase interaction being formed from the migration of early stage neural crest cell
With ultimately forming gingiva tissue.One typical feature of gingiva tissue is that have stronger wound healing ability, shows as cutting
Remove, damage after can no scar healing, recover gingival contour.
Gum mescenchymal stem cell (gingival mesenchymal stem cells, GMSCs) is composition gum connective
The main mesenchymal cell of tissue, from mesoblastic fibroblast, not only has the ability of active self, and
And also there is the function of the extracellular matrix such as synthesis and degraded collagen：I type and III Collagen Type VI, fibronectin splicing variants etc..Between gum
Mesenchymal stem cells draw materials conveniently, cell content is big in gingiva tissue, value-added speed is fast, as in adult mesenchymal stem cells
Member, is equally had using suitable derivant in vitro and is divided into cemental ability.This function of gum mescenchymal stem cell
Characteristic has greatly attracted to be derived from preclinical medicine and clinical medical researcher, provides one kind to the researchers of basic science
The biological pattern of research and development, reparation, replacement and the power of regeneration that gum mescenchymal stem cell has also is clinical medicine
Provide the chance of a development new treatment.Although there being the one-tenth using the Various Tissues source including fat, marrow for the research
Somatic stem cell carries out periodontal regenerative, but draw materials invasive and moral check the problems such as still constrain its development.Therefore, GMSCs
It is expected to become the seed cell of paradenlal tissue regeneration.
The research becoming fat induction currently for GMSCs focuses primarily upon different cytokines to its biological behaviour
The Different Effects producing, but often it is confined to the research of its effect of single cell factor pair.Because organism is a complexity
The many inducements of presence organism, therefore in the complicated microenvironment of periodontium, multiple inducible factors are in periodontal regenerative
During may play mutually collaborative or antagonism effect.Therefore it provides one kind has synergistic inducible factor group
Compound has important practical significance.
Content of the invention
In view of this, the invention provides a kind of adipogenic induction culture medium and become fat differentiation method.This adipogenic induction is cultivated
Base includes the inducible factors such as 3-isobutyl-1-methylxanthine, insulin, Indomethacin, dexamethasone, Rosiglitazone, PRP,
Multiple inducible factor use in conjunction act on the mescenchymal stem cells such as GMSCs, effectively fat can be become to divide by inducing mesenchymal stem cell
Change, it becomes fat differentiation effect to be significantly better than the abductive approach of existing routine.
In order to realize foregoing invention purpose, the present invention provides technical scheme below：
The invention provides a kind of adipogenic induction culture medium, including 3-isobutyl-1-methylxanthine, insulin, indoles
Mei Xin, dexamethasone, Rosiglitazone, PRP and basal medium.
In the adipogenic induction culture medium that the present invention provides, multiple inducible factor use in conjunction act on the mesenchymas such as GMSCs and do
Cell, the more efficiently one-tenth fat differentiation of the mescenchymal stem cell such as induction GMSCs.
Preferably, the consumption of each component is in adipogenic induction culture medium：
3-isobutyl-1-methylxanthine：0.05～0.15mmol/L；
Insulin：1～10mg/L；
Indomethacin：0.05～0.15mmoL/L；
Dexamethasone：0.5～1.5 μm of oL/L；
Rosiglitazone：0.1～1.0mmoL/L；
PRP：1%～10% volume fraction；
Basal medium：Supply.
Preferably, in adipogenic induction culture medium, the consumption of each component is：
3-isobutyl-1-methylxanthine：0.1mmol/L；
Insulin：5mg/L；
Indomethacin：0.1mmoL/L；
Dexamethasone：1μmoL/L；
Rosiglitazone：0.1～1.0mmoL/L；
PRP：1%～5% volume fraction；
Basal medium：Supply.
It is highly preferred that the consumption of each component is in adipogenic induction culture medium：
3-isobutyl-1-methylxanthine：0.1mmol/L；
Insulin：5mg/L；
Indomethacin：0.1mmoL/L；
Dexamethasone：1μmoL/L；
Rosiglitazone：0.5mmoL/L；
PRP：2.5% volume fraction；
Basal medium：Supply.
In the embodiment that the present invention provides, basal medium is the culture of low sugar DMEM containing 10% volume fraction FBS
Base.
Present invention also offers a kind of method that inducing mesenchymal stem cell becomes fat differentiation, including provided using the present invention
Adipogenic induction medium culture mescenchymal stem cell.
In the embodiment that the present invention provides, mescenchymal stem cell is gum mescenchymal stem cell.
Preferably, mescenchymal stem cell is the 2nd～5 generation mescenchymal stem cell.
Preferably, mescenchymal stem cell is the 3rd generation mescenchymal stem cell.
Preferably, the inoculum density of mescenchymal stem cell is (1～10) × 104cell/mL.
Preferably, the inoculum density of mescenchymal stem cell is 2 × 104cell/mL.
Preferably, the cultivation temperature of mescenchymal stem cell is 37 DEG C, incubation time is not less than 14 days.
Preferably, the incubation time of mescenchymal stem cell is not less than 21 days.
The invention provides a kind of adipogenic induction culture medium and one-tenth fat differentiation method.It is different that this adipogenic induction culture medium includes 3-
Butyl -1- methyl xanthine, insulin, Indomethacin, dexamethasone, Rosiglitazone, PRP and basal medium.The present invention is extremely
There is one of following advantage less：
1) present invention provides one kind to act on the mescenchymal stem cells such as GMSCs by multiple inducible factor use in conjunction, has
Synergy, effectively can become fat to break up by inducing mesenchymal stem cell, it becomes fat differentiation effect to be significantly better than luring of existing routine
Guiding method.
2) GMSCs autologous draw materials conveniently, be easy to amplification in vitro, can be used for autotransplantation, thus avoiding immunological rejection
Reaction.
Brief description
Fig. 1 shows GMSCs aspect graph；Wherein, 1-1 shows that GMSCs amplifies 40 times of pictures, and 1-2 shows that GMSCs amplifies 100 times of pictures；
Fig. 2 shows GMSCs cell surface marker flow cytometer detection result figure；Wherein, 2-1～2-6 shows CD146, HLA- respectively
The positive expression rate of DR, CD105, CD34, CD90, CD45；
Fig. 3 shows control group and experimental group cell oil red O stain design sketch；Wherein, 3-1～3-2 shows in control group 1 carefully respectively
Born of the same parents amplify 200 times, 400 times of picture after inducing 21 days；
3-3～3-4 shows that in control group 2, cell induction amplifies 200 times, 400 times of picture after 21 days respectively；
3-5～3-6 shows that in control group 3, cell induction amplifies 200 times, 400 times of picture after 21 days respectively；
3-7～3-8 shows that in control group 4, cell induction amplifies 200 times, 400 times of picture after 21 days respectively；
3-9～3-10 shows that in experimental group 1, cell induction amplifies 200 times, 400 times of picture in 21 days respectively；
3-11～3-12 shows that in experimental group 2, cell induction amplifies 200 times, 400 times of picture in 21 days respectively；
3-13～3-14 shows that in experimental group 3, cell induction amplifies 200 times, 400 times of picture in 21 days respectively；
Fig. 4 shows that control group and experimental group cell induction differentiation rate compare (p<0.01)；
Fig. 5 shows that the expression of the PPAR γ -2 and LPL of different time points control group and experimental group compares；Fig. 5-1 shows not
Expression with the PPAR γ -2 of time point control group and experimental group compares；Fig. 5-2 shows different time points control group and experiment
The LPL expression of group compares.
Specific embodiment
The invention discloses a kind of adipogenic induction culture medium and become fat differentiation method, those skilled in the art can use for reference this
Civilian content, is suitably modified technological parameter and realizes.Specifically, all similar replacements and change are to art technology
It is it will be apparent that they are considered as including in the present invention for personnel.The method of the present invention and application have been passed through preferably
Embodiment is described, and related personnel substantially can be to side as herein described in without departing from present invention, spirit and scope
Method and application are modified or suitably change and combine, and to realize and to apply the technology of the present invention.
Biomaterial used, reagent and instrument in the adipogenic induction culture medium of present invention offer and one-tenth fat differentiation method
Buied by market.
With reference to embodiment, the present invention is expanded on further：
Embodiment 1GMSCs is primary to be separately cultured, pass on and identifies
1st, GMSCs is primary is separately cultured
1) raw material sources：The gum that clinically orthodontic uprighting art cuts or impaction exodontia is taken to need and excise and surround perseverance
Periodontal gingiva tissue.Patient is required not have gingival hyperplasia, inflammation and the medicine leading to gingival hyperplasia using mistake.The tooth cutting
In the PBS containing 3 times of dual anti-4 DEG C precoolings for the quick immersion of gum tissue.
Wherein, 3 times of dual anti-concentration are 300u/mL penicillin, 300u/mL streptomysin.
2) rinse：Rinsed 3 times with the PBS dual anti-containing 3 times, then gum is placed in mesenchymal stem cell serum-free culture medium
Soak 5min；
Wherein, 3 times of dual anti-concentration are 300u/mL penicillin, 300u/mL streptomysin.
Wherein, 100u/mL penicillin, 100u/mL streptomysin are contained in mesenchymal stem cell serum-free culture medium.
3) digest for the first time：Gingiva tissue is cut into 1cm3Size, adds appropriate NTx enzyme-Dispase enzyme mixing to disappear
Change liquid, put 37 DEG C, in 200R constant-temperature table, digest 45min.After digestion terminates, 1000r/min is centrifuged 5min, abandons supernatant.Add
Appropriate PBS is resuspended, and 100um cell strainer filters suspension, the cell suspension 1000r/min centrifugation 5min after filtration, abandons supernatant.
Wherein, the ratio of NTx enzyme-Dispase enzyme is 1:1, collagenase concentration is clostridiopetidase A 3g/L, Dispase enzyme
Concentration is Dispase enzyme 4g/L.
Wherein, digestive environments are 37 DEG C, and in 200R constant-temperature table, digestion time is 45min；
Wherein, cell strainer aperture used is 100um；
Wherein, centrifugal speed is 1000r/min, and centrifugation time is 5min.
4) digest for second：Step 3) in filter under get off indigested tissue add isopyknic 0.125% pancreas
Protease, puts 37 DEG C, continues digestion 15min in 200R constant-temperature table.After digestion terminates, 1000r/min is centrifuged 5min, abandons
Clearly.Add appropriate PBS resuspended, 70um cell strainer filters suspension, the cell suspension 1000r/min centrifugation 5min after filtration, abandons
Supernatant.
Wherein, the ratio of trypsase and gingiva tissue is volume ratio 1:1, tryptic concentration is 0.125%；
Wherein, cell strainer aperture used is 70um；
Wherein, centrifugal speed is 1000r/min, and centrifugation time is 5min.
5) inoculate：Step 3) and step 4) in the cell collected with filling between EGF containing 10ng/mL (EGF)
Matter stem cell serum-free culture medium re-suspended cell, is inoculated in six orifice plates, the 37 DEG C of cultures of 5%CO2 incubator.
Wherein, basal medium is mesenchymal stem cell serum-free culture medium, can buy or make by oneself in market.
Wherein, in complete medium, the concentration of EGF (EGF) is 10ng/mL；
6) purify：Treat that cell covers with 80-90%, collect cell, with magnetic bead sorting method purifying cells.By collect cell with
4 DEG C of incubation 1h of STRO-1 monoclonal antibody, with 30min4 DEG C of incubation of magnetic bead, filter out STRO-1 under magnetic force devices effect+GMSCs, connects
Plant in six new orifice plates, 5%CO237 DEG C of cultures of incubator.
2nd, GMSCs passes on
Treat that cell covers with 80-90%, inhaled with suction pipe and abandon old nutrient solution, add 2-3mL0.25% trypsase, digest 1-3
Minute, Microscopic observation cellular contraction becomes bowlder, adds 5-10mL mesenchymal stem cell serum-free culture medium to terminate digestion immediately, receives
Collection cell, 1000r/min is centrifuged 5min, abandons supernatant.Add the mesenchymal stem cell serum-free training that 5-10mL contains 10ng/mL EGF
Foster base, carries out cell count, by 5 × 104Cell/mL density is inoculated in culture dish and carries out Secondary Culture, 5%CO2Incubator 37
DEG C culture, changed liquid every 2-3 days once.Observation of cell form in incubation, result is as shown in Figure 1.After cell inoculation about 4h
Start adherent, 3-4d enters exponential phase, 6-8d enters plateau.Vitro growth rates steadily, grow in monolayer adherence.Greatly
Part cell is in spindle shape, and form is irregular.
3rd, GMSCs surface marker identification
Treat that cell covers with 80-90%, collect cell (about 1 × 106Individual), add 3g/L Triton to soak 20min, PBS washes
3 times, 10% lowlenthal serum room temperature closing 2h, drip 1 respectively:CD146, CD105, CD90, CD34, CD45, HLA- of 200 dilutions
The each 50ul of DR antibody, the simple PBS 50ul of control group dropping simultaneously, 4 DEG C of process 16h.Next day room temperature places 30min, PBS rinsing 3
Time, each group drips 1 respectively:FITC mark two anti-igg of 500 dilutions, normal temperature lucifuge is incubated 1h, and PBS rinses 2 times, 10% Fu Er
Malin fixes cell, and flow cytometer measures the positive rate of antigen.Result of the test is shown in Table 1, Fig. 2.
Testing result shows, clone cell surface Antigens CD14 6, CD105, CD90 positive expression, and CD34, CD45,
The negative expression of HLA-DR, illustrates that the GMSCs obtaining in the present invention belongs to and derives from mesoblastic mescenchymal stem cell.
Table 1GMSCs cell surface marker expression rate result
| Cell phenotype | CD146 | CD105 | CD90 | HLA-DR | CD34 | CD45 |
| Positive expression rate (%) | 100.00 | 99.90 | 96.70 | 0.30 | 0.00 | 0.10 |
Embodiment 2GMSCs becomes fat differentiation
1st, the preparation of platelet rich plasma (PRP)
Collection and the peripheral blood of the same donor of tooth, proceed to 15mL centrifuge tube blood sample, 10mL often manages in super-clean bench,
2500rpm/min, is centrifuged 10min.Centrifugation terminates rear blood sample and is divided into faint yellow supernatant layer, middle leukocytic cream and red cell bottom
Layer.Draw whole supernatants, white cellular layer and red cell layer top 1-2mm, move into new 15mL centrifuge tube, 2500rpm/min,
Centrifugation 5min.Centrifugation draws supernatant layer and leukocytic cream after terminating, and moves into new 15mL centrifuge tube, 1200g/min, is centrifuged 5min.
Centrifugation abandons supernatant after terminating, and retains nethermost 2.5mL, i.e. platelet rich plasma.
2nd, PRP activation
The rush moving into chloride containing calcium coagulates pipe, and 4 DEG C overnight, 4000rpm/min, is centrifuged 12min, draws out complete in the solidifying pipe of rush
Portion's supernatant, 0.22 μm of filtration, -20 DEG C of preservations of filtrate are stand-by.
Test example
In order to verify that the present invention becomes the effect of fat induction, 4 groups of control groups and 3 groups of experimental group are set simultaneously, to experiment
Group carries out oil red O stain with control group and becomes aliphatic radical because of PPAR γ -2 and LPL detection of expression.
Control group 1 is negative control group, is cellar culture group, the nutrient solution of use is：Height containing volume fraction 10%FBS
Sugared DMEM nutrient solution.
Control group 2 is positive controls, fills a prescription for conventional mescenchymal stem cell adipogenic induction, and the adipogenic induction liquid of use is
0.1mmoL/L 3-isobutyl-1-methylxanthine (IBMX), 5mg/L insulin, 0.1mmoL/L Indomethacin, 1 μm of oL/L ground
Sai meter Song, volume fraction 10%FBS, balance of DMEM in high glucose nutrient solution.
Control group 3 is positive controls, and the adipogenic induction liquid of use is：0.1mmoL/L 3- isobutyl group -1- methyl yellow is fast
Purine (IBMX), 5mg/L insulin, 0.1mmoL/L Indomethacin, 1 μm of oL/L dexamethasone, 0.5mmoL/L Rosiglitazone, volume
Fraction 10%FBS, balance of DMEM in high glucose nutrient solution.
Control group 4 is positive controls, and the adipogenic induction liquid of use is：0.1mmoL/L 3- isobutyl group -1- methyl yellow is fast
Purine (IBMX), 5mg/L insulin, 0.1mmoL/L Indomethacin, 1 μm of oL/L dexamethasone, volume fraction 2.5%PRP, volume
Fraction 10%FBS, balance of DMEM in high glucose nutrient solution.
Used in experimental group 1, adipogenic induction liquid is：0.1mmoL/L 3-isobutyl-1-methylxanthine (IBMX),
5mg/L insulin, 0.1mmoL/L Indomethacin, 1 μm of oL/L dexamethasone, 0.1mmoL/L Rosiglitazone, volume fraction 1%
PRP, volume fraction 10%FBS, balance of DMEM in high glucose nutrient solution.
Used in experimental group 2, adipogenic induction liquid is：0.1mmoL/L 3-isobutyl-1-methylxanthine (IBMX),
5mg/L insulin, 0.1mmoL/L Indomethacin, 1 μm of oL/L dexamethasone, 0.5mmoL/L Rosiglitazone, volume fraction 2.5%
PRP, volume fraction 10%FBS, balance of DMEM in high glucose nutrient solution.
Used in experimental group 3, adipogenic induction liquid is：0.1mmoL/L 3-isobutyl-1-methylxanthine (IBMX),
5mg/L insulin, 0.1mmoL/L Indomethacin, 1 μm of oL/L dexamethasone, 1.0mmoL/L Rosiglitazone, volume fraction 5%
PRP, volume fraction 10%FBS, balance of DMEM in high glucose nutrient solution.
The formula of each group nutrient solution such as table 2：
The formula of table 2 each group nutrient solution
Effect example 1 oil red O stain
Take the 3rd generation GMSCs, by 2 × 104Cell/mL density is inoculated in 6 well culture plates, cultivates and reaches 80% to cell
When merging above, each group changes adipogenic induction liquid shown in table 2 respectively.Induction differentiation culture 21d carries out oil red O stain to cell.
Dyeing concrete grammar be：
Abandon nutrient solution, with PBS cell 2 to 3 times；4% paraformaldehyde is added to fix 15-20min；Poly first is abandoned in suction
Aldehyde, PBS 3 times；Add oil red O stain liquid, room temperature dyes 15-20min.Inhale after end and abandon raffinate, with going of 37 DEG C of preheatings
Ionized water cleans 2 to 3 times, each 20s；Observe under inverted microscope after drying and gather image.Each group cell dyeing effect
As Fig. 3.
The cell of oil red 0 dyeing is counted, the differentiation rate calculating two groups is compared.100 times of inverted microscope
Under magnification field, every hole counts at random 3 visuals field, calculates the ratio that noble cells accounts for TCS, parallel every time counts 3
Hole, calculates average).
Induction differentiation rate=staining positive cells number/TCS × 100%.
Each group induction differentiation rate result is as shown in table 3, Fig. 4.
Table 3 each group induces differentiation rate
Note：*, ※ represents P 0.01.
After result display culture 21d, control group 1 is negative control group, without inducible factor, does not carry out into fat to cell and divides
Change induction, therefore do not colour, become fat differentiation rate to be zero.Experimental group 1,3 and each control group all have significant difference (P
0.01, *)；Experimental group 2 and other each groups all have significant difference (P 0.01, * *)；One-tenth fat differentiation rate average in experimental group 2
For 86.27 ± 1.04%, it is 2.72 times of conventional induction differentiation group (control group 2).Illustrate that the differentiation of the one-tenth fat shown in the present invention lures
Drain becomes the effect of fat induction best GMSCs.
The detection of expression of effect example 2PPAR γ -2 and LPL
GMSCs is cultivated or is become according to experimental group or control group method with fat induction, continuous induction culture 14d and
After 21d, cell is carried out with the detection of expression of PPAR γ -2 and LPL.
Carry out cell total rna extraction by TRIzol kit specification；M-MLV reverse transcription reagent box obtains cDNA.Press
SYBR Green quantitative PCR kit specification detects to PPAR γ -2, LPL expression.Using GAPDH as internal reference base
Cause.Quantitative analysis is carried out with 2- △ △ Ct method.The primer using is as shown in table 4.Result is as shown in Figure 5.
Table 4 primer sequence table
Test result indicate that, after cell induction culture 14d, 21d, in same time point, each induction group is with not induce group (cloudy
Property control group) compare, lipoblast significant gene PPAR γ -2, LPL expression all have pole significant difference (*, P
0.01), experimental group 1,3 is compared with each control group, all has pole significant difference (※, p 0.01), experimental group 2 is respectively induced with other
Group all has pole significant difference (* *, p 0.01).
The above is only the preferred embodiment of the present invention it is noted that ordinary skill people for the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of adipogenic induction culture medium is it is characterised in that include 3-isobutyl-1-methylxanthine, insulin, indoles U.S.
Pungent, dexamethasone, Rosiglitazone, PRP and basal medium.
2. adipogenic induction culture medium according to claim 1 is it is characterised in that each component in described adipogenic induction culture medium
Consumption be：
3-isobutyl-1-methylxanthine：0.05～0.15mmol/L；
Insulin：1～10mg/L；
Indomethacin：0.05～0.15mmoL/L；
Dexamethasone：0.5～1.5 μm of oL/L；
Rosiglitazone：0.1～1.0mmoL/L；
PRP：1%～10% volume fraction；
Basal medium：Supply.
3. adipogenic induction culture medium according to claim 1 and 2 is it is characterised in that each in described adipogenic induction culture medium
The consumption of component is：
3-isobutyl-1-methylxanthine：0.1mmol/L；
Insulin：5mg/L；
Indomethacin：0.1mmoL/L；
Dexamethasone：1μmoL/L；
Rosiglitazone：0.1～1.0mmoL/L；
PRP：1%～5% volume fraction；
Basal medium：Supply.
4. adipogenic induction culture medium according to any one of claim 1 to 3 is it is characterised in that described adipogenic induction is trained
In foster base, the consumption of each component is：
3-isobutyl-1-methylxanthine：0.1mmol/L；
Insulin：5mg/L；
Indomethacin：0.1mmoL/L；
Dexamethasone：1μmoL/L；
Rosiglitazone：0.5mmoL/L；
PRP：2.5% volume fraction；
Basal medium：Supply.
5. adipogenic induction culture medium according to any one of claim 1 to 4 is it is characterised in that described basal medium
It is the low sugar DMEM culture medium containing 10% volume fraction FBS.
6. a kind of inducing mesenchymal stem cell becomes the method for fat differentiation it is characterised in that adopting any one of claim 1 to 5
Adipogenic induction medium culture mescenchymal stem cell.
7. method according to claim 6 is it is characterised in that described mescenchymal stem cell is gum mescenchymal stem cell.
8. the method according to claim 6 or 7 is it is characterised in that described mescenchymal stem cell is the 2nd～5 generation mesenchyma
Stem cell.
9. the method according to any one of claim 6 to 8 is it is characterised in that the inoculation of described mescenchymal stem cell is close
Spend for (1～10) × 104cell/mL.
10. the method according to any one of claim 6 to 9 is it is characterised in that the culture temperature of described mescenchymal stem cell
Spend for 37 DEG C, incubation time is not less than 14 days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610906762.7A CN106381283A (en) | 2016-10-18 | 2016-10-18 | Adipogenesis induction culture medium and adipogenic differentiation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610906762.7A CN106381283A (en) | 2016-10-18 | 2016-10-18 | Adipogenesis induction culture medium and adipogenic differentiation method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106381283A true CN106381283A (en) | 2017-02-08 |
Family
ID=57958745
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610906762.7A Pending CN106381283A (en) | 2016-10-18 | 2016-10-18 | Adipogenesis induction culture medium and adipogenic differentiation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106381283A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108888633A (en) * | 2018-09-21 | 2018-11-27 | 北京泰盛生物科技有限公司 | A kind of gum mescenchymal stem cell preparation and its application and preparation method |
| CN109679901A (en) * | 2019-02-21 | 2019-04-26 | 山东华思生物科技有限公司 | A kind of adipogenic induction differentiation culture solution and adipogenic induction differentiation method |
| CN109694849A (en) * | 2018-12-28 | 2019-04-30 | 山西医科大学第一医院 | Culture medium and method of the rapid induction human marrow mesenchymal stem cell to Adipose Differentiation |
| CN114181899A (en) * | 2021-12-22 | 2022-03-15 | 中山大学孙逸仙纪念医院 | Method for obtaining mesenchymal stem cells from gum tissue of mice |
| CN115232786A (en) * | 2022-08-02 | 2022-10-25 | 莫伊森(厦门)生发有限公司 | Adipose-derived stem cell exosome and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011124741A1 (en) * | 2010-04-08 | 2011-10-13 | Fundación Progreso Y Salud | Use of a culture medium conditioned by mesenchymal stem cells for differentiation of pluripotent human stem cells |
| CN104837988A (en) * | 2012-11-30 | 2015-08-12 | 瑞士干细胞基金会 | Serum-free medium for human mesenchymal stem cells |
| CN104830757A (en) * | 2015-04-15 | 2015-08-12 | 广州赛莱拉干细胞科技股份有限公司 | Mesenchymal stem cell adipogenic induced differentiation culture medium and preparation method thereof |
| CN105462917A (en) * | 2015-11-30 | 2016-04-06 | 广州赛莱拉干细胞科技股份有限公司 | Periodontal ligament stem cell adipogenic differentiation inducing liquid and method |
-
2016
- 2016-10-18 CN CN201610906762.7A patent/CN106381283A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011124741A1 (en) * | 2010-04-08 | 2011-10-13 | Fundación Progreso Y Salud | Use of a culture medium conditioned by mesenchymal stem cells for differentiation of pluripotent human stem cells |
| CN104837988A (en) * | 2012-11-30 | 2015-08-12 | 瑞士干细胞基金会 | Serum-free medium for human mesenchymal stem cells |
| CN104830757A (en) * | 2015-04-15 | 2015-08-12 | 广州赛莱拉干细胞科技股份有限公司 | Mesenchymal stem cell adipogenic induced differentiation culture medium and preparation method thereof |
| CN105462917A (en) * | 2015-11-30 | 2016-04-06 | 广州赛莱拉干细胞科技股份有限公司 | Periodontal ligament stem cell adipogenic differentiation inducing liquid and method |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108888633A (en) * | 2018-09-21 | 2018-11-27 | 北京泰盛生物科技有限公司 | A kind of gum mescenchymal stem cell preparation and its application and preparation method |
| CN109694849A (en) * | 2018-12-28 | 2019-04-30 | 山西医科大学第一医院 | Culture medium and method of the rapid induction human marrow mesenchymal stem cell to Adipose Differentiation |
| CN109679901A (en) * | 2019-02-21 | 2019-04-26 | 山东华思生物科技有限公司 | A kind of adipogenic induction differentiation culture solution and adipogenic induction differentiation method |
| CN109679901B (en) * | 2019-02-21 | 2022-06-24 | 山东华思生物科技有限公司 | Adipogenic induced differentiation culture solution and adipogenic induced differentiation method |
| CN114181899A (en) * | 2021-12-22 | 2022-03-15 | 中山大学孙逸仙纪念医院 | Method for obtaining mesenchymal stem cells from gum tissue of mice |
| CN115232786A (en) * | 2022-08-02 | 2022-10-25 | 莫伊森(厦门)生发有限公司 | Adipose-derived stem cell exosome and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106381283A (en) | Adipogenesis induction culture medium and adipogenic differentiation method | |
| Yu et al. | LPS‑induced upregulation of the TLR4 signaling pathway inhibits osteogenic differentiation of human periodontal ligament stem cells under inflammatory conditions | |
| KR101328604B1 (en) | Autologous and allogenic adipose tissue-derived stromal stem cells composition for treatment of fistula | |
| CN106434539A (en) | Osteogenic induction medium and osteogenic differentiation method | |
| CN106906182A (en) | A kind of mesenchymal stem cell serum-free culture medium | |
| CN106318904A (en) | Use of mesenchymal stem cell conditioned culture medium for cosmetics field | |
| CN104762257B (en) | A kind of method preparing mescenchymal stem cell from umbilical cord | |
| US20130029292A1 (en) | Method of implanting mesenchymal stem cells for natural tooth regeneration in surgically prepared extraction socket and compositions thereof | |
| CN103301154B (en) | Application of umbilical cord mesenchymal stem cells in preparation of formulation for treating lupus erythematosus | |
| CN109234229A (en) | Method and digestive enzyme compositions used from placenta blood vessel separating mesenchymal stem cell | |
| CN102191217A (en) | Method for inducing differentiation from human umbilical cord mesenchymai stem cells (hucMSCs) into neural cells | |
| CN106929470A (en) | It is a kind of for derived mesenchymal stem cells in vitro culture and amplification serum free medium | |
| CN106244533A (en) | The primary separation method of gingiva mescenchymal stem cell | |
| Zhang et al. | Human umbilical cord mesenchymal stem cell differentiation into odontoblast-like cells and endothelial cells: a potential cell source for dental pulp tissue engineering | |
| CN105238746A (en) | Inducing method and inducing liquid of mesenchymal stem cells | |
| CN105255824A (en) | Periodontal ligament stem cell osteogenic differentiation inducing liquid and method | |
| JP5388297B2 (en) | Adipo cluster | |
| CN107287156A (en) | A kind of isolated culture method of fat mesenchymal stem cell and its application | |
| CN110475855A (en) | For the method that cell prepares dental pulp stem cell derived from pulp tissue | |
| CN104031881B (en) | A kind of isolated culture method of mankind's olfactory mucosa mescenchymal stem cell | |
| CN116640727B (en) | Nutrient solution for improving cell viability and preparation method and application thereof | |
| CN108865985A (en) | A kind of method of the pre- epithelial-mesenchymal conversion of stem cell source excretion soma | |
| CN109628388A (en) | With digestive enzyme compositions from placenta blood vessel separating mesenchymal stem cell | |
| Salvadè et al. | GMP-grade preparation of biomimetic scaffolds with osteo-differentiated autologous mesenchymal stromal cells for the treatment of alveolar bone resorption in periodontal disease | |
| CN106834217A (en) | A kind of method for promoting human amnion membrane amplification in vitro and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170208 |